CN103823014B - Compositionss, test kit and application thereof and with its analysis of amino acid method - Google Patents
Compositionss, test kit and application thereof and with its analysis of amino acid method Download PDFInfo
- Publication number
- CN103823014B CN103823014B CN201410048846.2A CN201410048846A CN103823014B CN 103823014 B CN103823014 B CN 103823014B CN 201410048846 A CN201410048846 A CN 201410048846A CN 103823014 B CN103823014 B CN 103823014B
- Authority
- CN
- China
- Prior art keywords
- acid
- alcohol
- water
- aminoacid
- container
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Active
Links
Landscapes
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The present invention provides a kind of compositionss, and which includes the mixture of alcohol, acid and water and 1,2,3-indantrione monohydrate, and the present invention also provides a kind of test kit, and which includes to accommodate containing alcohol, acid or the one of which of water, two or three of container and accommodates the container containing 1,2,3-indantrione monohydrate.Present invention simultaneously provides the method that said composition and test kit are used for the purposes and analysis of amino acid of analysis of amino acid, methods described can use the compositionss or test kit of the present invention carry out.During aminoacid in compositionss and test kit in the analysis of amino acid and particularly fermentation liquid of the present invention, color rendering propertiess are good, high resolution, aminoacid in can analyzing at least 13.The method of the present invention not only low cost, it is easy to operate, and detect fast, it is only necessary to and 10 25 minutes, additionally, the method for the present invention can analyze at least 13 kinds aminoacid high-resolution.
Description
Technical field
The present invention relates to a kind of compositionss and test kit, which is used for analysis of amino acid, aminoacid particularly in fermentation liquid
Purposes, further relates to the method with aminoacid in its analysis of amino acid, particularly fermentation liquid.
Background technology
At present, analyze the Amino Acid Composition in fermentation liquid and mainly use following methods：1）High performance liquid chromatography or aminoacid
Analyser, although both approaches can carry out quantitative analyses, instrument is expensive, it is difficult to popularize；2）Paper chromatography,
Although this method is not high to equipment requirements, also relatively facilitate, it is long that this method chromatographs the time every time, it usually needs 2-3 hours, always
Analysis time is then up to 4 hours, and traditional paper chromatography is developed the color using spraying, and easily colour developing is uneven or breaks up mottle, affects to divide
Analysis judges.
Zhao Jianguo etc. is in document《Thin layer chromatography separates the research of protein and Amino acids in stiff Cicadae albumen》, lake
Southern college of traditional Chinese medicine's report, in April, 2005, volume 25, the 2nd phase：The 26-29 page disclose a kind of stiff Cicadae protein and amino acidses into
The TLC separation discrimination method for dividing, although the method can realize the analysis of stiff Cicadae Amino Acids in Proteins, but when launching
Between need 90 minutes, the bulk analysis time is longer, and the limitednumber of analyzable aminoacid.
The content of the invention
The present invention provides a kind of compositionss, and the compositionss are the mixture containing alcohol, acid, water and 1,2,3-indantrione monohydrate.Described group
Compound can be used for analysis of amino acid, the aminoacid being particularly in fermentation liquid.
In an embodiment of the present composition, wherein the mixture containing alcohol, acid, water and 1,2,3-indantrione monohydrate by alcohol,
Acid, water and 1,2,3-indantrione monohydrate are formulated, the alcohol be n-butyl alcohol and one or more selected from methanol, ethanol, normal propyl alcohol, isopropanol,
Tertiary propanol, isobutanol, the tert-butyl alcohol, n-amyl alcohol, the alcohol mixture of isoamyl alcohol.In a preferred embodiment of the present composition
In, the alcohol is the alcohol mixture of n-butyl alcohol and ethanol.In a preferred embodiment of the present composition, the alcohol is mixed
Compound mixes mol ratio 1/1-5/1.
In a preferred embodiment of the present composition, wherein the mixture containing alcohol, acid, water and 1,2,3-indantrione monohydrate by
Alcohol, acid, water and 1,2,3-indantrione monohydrate are formulated.In a preferred embodiment of the present composition, wherein containing alcohol, acid, water
It is formulated by alcohol, acid, water and solid 1,2,3-indantrione monohydrate with the mixture of 1,2,3-indantrione monohydrate.
In a preferred embodiment of the present composition, wherein acid selected from formic acid, acetic acid, propanoic acid, butanoic acid, penta
Acid, glacial acetic acid.In a more preferred of the present composition, acid is glacial acetic acid.
In a preferred embodiment of the present composition, the amount of alcohol is 20-50 mole of %, and the amount of acid is rubbed for 10-35
You are %, and the amount of water is 30-60 mole of %, and the amount sum based on the alcohol, acid and water is 100 moles of % meters；The amount of 1,2,3-indantrione monohydrate is per 1
Rise 0.2-10 gram of solvent.
The present invention also provides a kind of test kit, and the test kit is included selected from one of following container：
Accommodate the container containing alcohol, acid, water and 1,2,3-indantrione monohydrate mixture；
Accommodate containing alcohol, the container of acid and water and accommodate the container containing 1,2,3-indantrione monohydrate；Or
Accommodated containing alcohol, acid, the container of water respectively and accommodate the container containing 1,2,3-indantrione monohydrate；Or
The container of optional two kinds in being accommodated containing alcohol, acid, water respectively and containing another material in alcohol, acid, water
The container of container and receiving containing 1,2,3-indantrione monohydrate,
The alcohol is that n-butyl alcohol is selected from methanol, ethanol, normal propyl alcohol, isopropanol, tertiary propanol, isobutanol, tertiary fourth with any one
Alcohol, n-amyl alcohol, the alcohol mixture of isoamyl alcohol.In a preferred embodiment of test kit of the present invention, the alcohol is n-butyl alcohol
With the alcohol mixture of ethanol.In a preferred embodiment of test kit of the present invention, the mixing mol ratio of the alcohol mixture
1/1-5/1。
In a preferred embodiment of test kit of the present invention, wherein the mixture containing alcohol, acid, water and 1,2,3-indantrione monohydrate by
Alcohol, acid, water and solid 1,2,3-indantrione monohydrate are formulated；Or, what is accommodated in the container of the receiving containing 1,2,3-indantrione monohydrate is solid indenes three
Ketone.
In a preferred embodiment of test kit of the present invention, in the test kit of the present invention, also include flat board, which is by moulding
Material, glass, ceramics etc. are constituted.
In a further preferred embodiment of test kit of the present invention, the test kit of the present invention is also included containing control sample
Container.
In the various embodiments of test kit of the present invention, the description previously for each preferred embodiment of compositionss is same
It is suitable for, and constitutes corresponding preferred, further preferred embodiment.
The present invention also provides the purposes that the compositionss or test kit of the present invention are used for analysis of amino acid.
In a preferred embodiment of purposes of the present invention, wherein aminoacid to be analyzed is the amino in fermentation liquid
Acid.
In the preferred embodiment of purposes of the present invention, fermentation liquid therein is to obtain after fermented product dilute with water
Aqueous solution.
In a more preferred of purposes of the present invention, fermentation liquid is obtained for 1-100 times of fermented product dilute with water
Fermentation liquid.
In a preferred embodiment of purposes of the present invention, wherein fermented product is with selected from glucose, Lactose, sucrose, wheat
The sugar of bud sugar, Semen Maydis oil, amylum hydrolysate of the sugar etc. is for substrate and with selected from Deshi Lactobacilluss（Lactobacillus
delbrueckii）, lactobacillus delbruockii subspecies bulgaricus, Deshi Lactobacilluss Lai Ku South Asia kind（L.leichnanii）Micro- life
Thing carries out cultivation and fermentation acquisition.
In each embodiment of purposes of the present invention, the aminoacid includes lysine, aspartic acid, glutamic acid, egg ammonia
Acid, isoleucine, arginine, histidine, alanine, tryptophan, L-Tyrosine, serine, Phenylalanine, threonine.
Additionally, the present invention also provides a kind of method of analysis of amino acid, methods described comprises the steps：
A) point sample：By the sample spot containing aminoacid on flat board；
B) open up layer-colour developing：One end that point has the flat board containing aminoacid sample is placed in into containing alcohol, acid, water and 1,2,3-indantrione monohydrate
In mixture,
The mixture of containing alcohol described in step b), acid and water and 1,2,3-indantrione monohydrate derives from the examination of the compositionss or the present invention of the present invention
Agent box.
In a preferred embodiment of the inventive method, the method for the present invention also including step c) treat the containing alcohol,
After the completion of sour, water and 1,2,3-indantrione monohydrate mixture opens up layer-colour developing on the flat board, flat board is taken out, which is dried, it is seen that
The speckle of various aminoacid.
In a preferred embodiment of the inventive method, the method for the analysis of amino acid of the present invention also includes d) inciting somebody to action
Compare from flat board c) with the flat board of standard sample.
In a preferred embodiment of the inventive method, the flat board in step a) is lamellae.
In a preferred embodiment of the inventive method, in step a), the sample containing aminoacid is aminoacid standard
Sample and/or its mixture, and the solution containing aminoacid.
In a more preferred of the inventive method, wherein the solution containing aminoacid is containing aminoacid
Fermentation liquid.
In the more preferred of the inventive method, wherein fermentation liquid is to obtain after fermented product dilute with water
Aqueous solution.
In one of the inventive method even more preferably embodiment, wherein fermentation liquid is 1-100 times of dilute with water
The fermentation liquid for arriving.
In a preferred embodiment of the inventive method, wherein fermented product is with selected from glucose, Lactose, sucrose, wheat
Bud sugar, Semen Maydis oil, starch（Such as potato starch, corn starch, tapioca）The sugar of hydrolysis sugar is for substrate and with selected from moral
Family name's lactobacilluss（Lactobacillus delbrueckii）, lactobacillus delbruockii subspecies bulgaricus, Deshi Lactobacilluss Lai Ku South Asia
Kind（L.leichnanii）Microorganism carry out cultivation and fermentation acquisition.
In a preferred embodiment of the inventive method, the point sample step in step a) is repeatedly carried out.
In a more preferred of the inventive method, after the completion of each point sample, also include the step being dried
Suddenly.
In a preferred embodiment of the inventive method, in step b), the liquid level of the present composition is not higher than flat board
On sample spot.
In each embodiment of the inventive method, the aminoacid includes lysine, aspartic acid, glutamic acid, egg ammonia
Acid, isoleucine, arginine, histidine, alanine, tryptophan, L-Tyrosine, serine, Phenylalanine, threonine.
During aminoacid in compositionss and test kit in the analysis of amino acid and particularly fermentation liquid of the present invention, show
Color is good, high resolution, can analyze at least 13 kinds aminoacid.
The method of the present invention not only low cost, it is easy to operate, and detect fast, it is only necessary to and 10-25 minutes, additionally, of the invention
Method can analyze at least 13 kinds aminoacid high-resolution.
Specific embodiment
The present invention provides a kind of compositionss, and which includes：Mixture containing alcohol, acid, water and 1,2,3-indantrione monohydrate.
The available alcohol of the present composition can be commercially available known alcohol, such as methanol, ethanol, normal propyl alcohol, isopropanol, uncle
Propanol, n-butyl alcohol, isobutanol, the tert-butyl alcohol, n-amyl alcohol, isoamyl alcohol and/or its mixture, but not limited to this.The present composition
The preferred n-butyl alcohol of available alcohol and any one selected from methanol, ethanol, normal propyl alcohol, isopropanol, tertiary propanol, isobutanol, the tert-butyl alcohol, just
The alcohol mixture of the alcohol mixture of amylalcohol, isoamyl alcohol, more preferably n-butyl alcohol and ethanol, in the case of alcohol mixture, the alcohol
The mixing mol ratio of mixture be 1/1-5/1, preferably 3/2-4/1, more preferably 2/1-7/2.
The available acid of the present composition can be commercially available known acid, for example formic acid, acetic acid, propanoic acid, butanoic acid, valeric acid,
Glacial acetic acid, but not limited to this.The available acid of the present composition preferably acetic acid, glacial acetic acid, more preferably glacial acetic acid.
The available water of the present composition can be, such as deionized water, but not limited to this.
The available 1,2,3-indantrione monohydrate of the present composition can be commercially available prod, such as purchased from the limited public affairs of Chinese favour generation biochemical reagents
The pure 1,2,3-indantrione monohydrate of analysis of department, purchased from the pure 1,2,3-indantrione monohydrate of analysis of Shanghai one bio tech ltd of China, but not limited to this.
In an embodiment of the present composition, the compositionss of the present invention are containing alcohol, acid, water and 1,2,3-indantrione monohydrate
Mixture, which is formulated by alcohol, acid, water and 1,2,3-indantrione monohydrate.The 1,2,3-indantrione monohydrate can be solid 1,2,3-indantrione monohydrate.
In the compositionss of the present invention, the amount of alcohol is 20-50 mole of %, preferred 25-45 mole of %, more preferably 30-40 mole %；
The amount of acid is 10-35 mole of %, preferred 15-30 mole of %, more preferably 18-25 mole %；The amount of water is 30-60 mole of %, preferred 35-
50 moles of %, more preferably 40-45 mole %；Amount sum based on the alcohol, acid and water is 100 moles of % meters.The compositionss of the present invention
In, the amount of 1,2,3-indantrione monohydrate is per 0.2-10 gram, preferred 0.5-8 gram, more preferably 0.8-5 gram of 1 liter of solution.The amount of the 1,2,3-indantrione monohydrate is pure
Material（Non-hydrate）Amount, it should be appreciated that available 1,2,3-indantrione monohydrate of the invention both include its pure material, also including its hydrate or its
His material form, for the hydrate or other material forms of 1,2,3-indantrione monohydrate, the amount should be scaled the amount of pure 1,2,3-indantrione monohydrate pure material.
The present invention also provides a kind of test kit, and which is included selected from one of following container：
Accommodate the container containing alcohol, acid, water and 1,2,3-indantrione monohydrate mixture；
Accommodate containing alcohol, the container of acid and water and accommodate the container containing 1,2,3-indantrione monohydrate；Or
Accommodated containing alcohol, acid, the container of water respectively and accommodate the container containing 1,2,3-indantrione monohydrate；Or
The container of optional two kinds in being accommodated containing alcohol, acid, water respectively and containing another material in alcohol, acid, water
The container of container and receiving containing 1,2,3-indantrione monohydrate.
The available alcohol of test kit of the present invention and its amount can be the above-mentioned alcohol referred to for the present composition and its amount.
Test kit of the present invention is available sour and its amount can be the above-mentioned acid referred to for the present composition and its amount.
The available water of test kit of the present invention and its amount can be the above-mentioned water referred to for the present composition and its amount.
The available 1,2,3-indantrione monohydrate of test kit of the present invention and its amount can be the above-mentioned 1,2,3-indantrione monohydrates referred to for the present composition
And its amount.
In a preferred embodiment of test kit of the present invention, the mixture containing alcohol, acid, water and 1,2,3-indantrione monohydrate by
Alcohol, acid, water and solid 1,2,3-indantrione monohydrate are formulated；Or, what is accommodated in the container of the receiving containing 1,2,3-indantrione monohydrate is solid indenes three
Ketone.
Herein, accommodate the container containing alcohol, acid, water and 1,2,3-indantrione monohydrate mixture be 1 container, its accommodate by alcohol,
The mixture that acid, water and 1,2,3-indantrione monohydrate are formulated；Accommodate containing alcohol, acid and water container be 1 container, its accommodate containing
The mixture of alcohol, acid and water；The container containing alcohol, acid, water is accommodated respectively for 3 containers, this 3 containers accommodate respectively containing
Alcohol, containing acid and the material containing water；The container of optional two kinds in being accommodated containing alcohol, acid, water respectively and contain alcohol, acid, water
In the container of another material be 2 containers, wherein first container accommodate alcohol, acid, any two kinds of mixture in water,
Second container accommodates alcohol, acid, the material being not accommodated in first container in water；It is 1 to accommodate the container containing 1,2,3-indantrione monohydrate
Individual container, which accommodates the material containing 1,2,3-indantrione monohydrate, and the material containing 1,2,3-indantrione monohydrate can be such as 1,2,3-indantrione monohydrate solid, indenes three
The hydrate of ketone, solution of 1,2,3-indantrione monohydrate etc..
It should be understood that it is exemplary only with the quantity of upper container and receiving mode, as long as including present composition component
Alcohol, acid, the container of water and 1,2,3-indantrione monohydrate, no matter the number of container and accommodate mode, within the scope of the present invention.
The capacity of container, the container of the container of preferred low capacity, such as capacity for 10-25 milliliters are not limited.
Container for example can be made up of plastics or glass, such as by polyethylene terephthalate（PET）Make, for example, be purchased from
The 15mlPET bottles of Taizhou Ping Jisu industry company limited.The container cross section is not limited, for example, can be circle, for example container can
For right cylindrical.The container can be transparent, translucent or opaque；Container can have color, such as amber, for example, purchase
From the 15ml brown reagent bottle of Hangzhou Lai Baisi experimental facilitiess company limited.
The form of container is not limited, can be bottle, bag, bag, medicated bag or tank.
What container was preferably closed.
To dispense and selling, container can be provided in outer package such as carton.
Use for convenience, flat board in the test kit of the present invention, can also be included, which is made up of plastics, glass, ceramics etc..
It should be understood that flat board can be lamellae, lamellae is not limited in any way, can be any commercially available detection lamellae, example
Such as purchased from General experimental science and technology（Hong Kong）Company limitedLamellae, purchased from MERCK MILLIPORE（Merck
Mi Libo）Thin layer chromatography board, purchased from City of Taizhou road and bridge tetramethyl biochemistry plastic molding and processing plant GF254- chromatographic silica gel thin plates.
Available flat board in the present invention is light color, preferred white.
The container containing control sample can also be included in the test kit of the present invention, the control sample could be for contrasting
Go out the single pure sample or the mixture of pure sample of result, for example, it may be one group of known amino acid pure sample product.It should be understood that described
Control sample is placed in different containers from the component of the present composition.Additionally, control sample can also be with the combination of the present invention
Thing is provided in different cartons, i.e., compositionss of the invention can be included in sale in a box simultaneously with control sample, also may be used
Sold with being separately contained in different boxes.
The present invention also provides the purposes that the compositionss or test kit of the present invention are used for analysis of amino acid.
The present invention also provides the compositionss or test kit of the present invention to be used to analyze the purposes of aminoacid in fermentation liquid.
Analyzable aminoacid refers to the organic acid containing amino in its molecular formula.Aminoacid can for example rely ammonia
Acid, aspartic acid, glutamic acid, methionine, isoleucine, arginine, histidine, alanine, tryptophan, L-Tyrosine, serine,
Phenylalanine and threonine.Herein, aminoacid can be in the form or the form in corresponding salt of acid.
The fermentation liquid being mentioned above is fermented product water, the solution that preferred deionized water is obtained after diluting, and dilution ratio is
1-100 times, preferred 10-80 times, more preferably 15-50 times.
The fermented product being mentioned above generally carries out cultivation and fermentation acquisition by substrate of sugar with microorganism, and the sugar can be with Fructus Vitis viniferae
Sugar, Lactose, sucrose, maltose, Semen Maydis oil, starch（Such as potato starch, corn starch, tapioca）Hydrolysis sugar, it is described
Microorganism can be, such as Deshi Lactobacilluss（Lactobacillus delbrueckii）, Lactobacillus delbrueckii it is sub-
Kind, Deshi Lactobacilluss Lai Ku South Asia kind（L.leichnanii）.
In an embodiment of purposes of the present invention, with the compositionss or kit assay of the present invention with selected from Fructus Vitis viniferae
Sugar, Lactose, sucrose, maltose, Semen Maydis oil, starch（Such as potato starch, corn starch, tapioca）The sugar of hydrolysis sugar is
Substrate with selected from Deshi Lactobacilluss（Lactobacillus delbrueckii）, lactobacillus delbruockii subspecies bulgaricus, De Shi
Lactobacilluss Lai Ku South Asia kind（L.leichnanii）Microorganism carry out 1-100 times of the fermented product dilute with water of cultivation and fermentation acquisition
Solution.
In a preferred embodiment of purposes of the present invention, with the compositionss or kit assay of the present invention with glucose
For substrate and with Deshi Lactobacilluss（Lactobacillus delbrueckii）The fermented product for carrying out cultivation and fermentation acquisition is dilute with water
Release 10-80 times of solution.
In a more preferred of purposes of the present invention, with the compositionss or kit assay of the present invention with Fructus Vitis viniferae
Sugar is for substrate and with Deshi Lactobacilluss（Lactobacillus delbrueckii）Carry out the fermented product water of cultivation and fermentation acquisition
The solution of 15-50 times of dilution.
Additionally, the present invention also provides a kind of method of analysis of amino acid, methods described comprises the steps：
A) point sample：By the sample spot containing aminoacid on flat board；
B) open up layer-colour developing：One end that point is had the flat board of the sample spot containing aminoacid is placed in containing alcohol, acid, water and indenes three
In the mixture of ketone.
Sample containing aminoacid can be aminoacid standard sample or its mixture, be selected from lysine, Radix Asparagi ammonia
Acid, glutamic acid, methionine, isoleucine, arginine, histidine, alanine, tryptophan, L-Tyrosine, serine, Phenylalanine
With the aminoacid and its mixture, or the solution containing aminoacid of threonine, for example, can be sending out containing aminoacid
Zymotic fluid.
In step a), different according to the concentration containing aminoacid sample, point sample can be carried out by several times, such as 1-10 time, excellent
2-8 point sample of choosing point, more preferably divides 3-5 point sample.Preferably, in the case of gradation point sample, carry out after the completion of each point sample
It is dried.Drying can be carried out under room temperature or heating, be such as thermally dried, and can use any of con-ventional drying equipment,
Can for example be drying baker, hair-dryer.
In step a), point sample can be carried out with any of liquid shifting equipment, for example, can be that by present invention side
Any liquid shifting equipment of the method step a) purposes, such as pipet, microsyringe.
In step b), the liquid level of the preferred present composition is not higher than the sample spot on flat board.
In step b), point has the time that the flat board containing aminoacid sample is placed in the present composition to be not particularly limited,
As long as completing to open up layer-colour developing, in other words, the compositionss of the present invention are along the one end for the flat board being placed in one to other end exhibition
The edge of layer to the other end, the time can be, such as 8-50 minutes, preferred 10-30 minutes, more preferably 10-25
Minute.
In step b), the present invention and 1,2,3-indantrione monohydrate mixture can be by the compositionss of the present invention or the test kit of the present invention
There is provided.
In a preferred embodiment of the inventive method, also including c) treat the present invention compositionss on the flat board
After the completion of exhibition layer-colour developing, flat board is taken out, which is dried, it is seen that the speckle of various aminoacid.
In step c), drying can be carried out at normal temperatures, it is also possible to carry out under heating, preferably carry out under heating, plus
The temperature of heated drying can be 30 DEG C -80 DEG C, and preferred 35-75 DEG C, drying time is not particularly limited, as long as can realize drying mesh
, can be adjusted according to drying temperature, for example, dry such as at 70 DEG C, can be 2-5 minutes.
In a preferred embodiment of the inventive method, also including d) flat board for deriving from c) is compared with standard sample.
As a result calculate
During with lamellae analysis of amino acid, relative mobility Rf of the aminoacid on lamellae is generally used（rate of
flow）To carry out discriminating separation to which.Specifically, Rf value is the distance that aminoacid is migrated along the solvent direction of motion（d1）With ammonia
Base acid when not migrating apart from liquid level distance（d2）Ratio.
Rf=d1/d2
Herein, for example, Rf can be measured by the following manner：The step of said method in b), have containing amino in point
When one end of the flat board of sour sample is placed in the compositionss of the present invention, the sample spot containing aminoacid is measured（Origin）Centre distance
Present composition liquid level apart from d2；Measure on the flat board for deriving from above-mentioned analysis method step b) Jing after exhibition layer-colour developing
Aminoacid spot centers are to origin center apart from d1, the Rf of aminoacid in fermentation liquid described herein is calculated according to equation below
Value.It is the solution containing aminoacid such as to analyze object, then according to above-mentioned calculated Rf value and known standard amino acid sample spot
RfMarkValue is compared, and determines the species of aminoacid in fermentation liquid of the present invention, the Rf according to comparative resultMarkThe survey of value
Surely can carry out in the method in accordance with the invention.
The preferred embodiment of the present invention will be described in detail by embodiment below, it should be appreciated that following examples are only to show
Example property, should not be construed as limiting the scope of the invention.
Embodiment 1：Prepare the compositionss of the present invention
Raw material
Ethanol, top pure grade, Beijing Chemical Plant
N-butyl alcohol, top pure grade, Beijing Chemical Plant
Glacial acetic acid, top pure grade, Beijing Chemical Plant
Deionized water, self-control
1,2,3-indantrione monohydrate, analyzes pure, Chinese Hui Shi biochemical reagents company limited
The standard sample of aminoacid, Chinese Hui Shi biochemical reagents company limited
Experiment equipment
Chromatographic silica gel thin plate, 5cm × 3cm, GF254- thin plate, City of Taizhou road and bridge tetramethyl biochemistry plastic molding and processing plant
Ruler, pencil, it is commercially available
Hair-dryer, Philip 1600w
Chromatography cylinder, 7cm × 7cm × 10cm, Tianjin Glass Instrument Factory
Baking oven, 202-0 table electrothermal drying baker, Tianjin Stettlen Instrument Ltd.
1 microlitre of microsyringe, YY0088-92, the love instrument plant of Ningbo City Zhenghai three
Prepare fermentation liquid
With glucose as substrate, submerged fermentation being carried out from moral formula lactobacilluss, obtaining fermented product, deionized water will be above-mentioned
Fermented product dilutes 25 times, obtains fermentation liquid.
Prepare the compositionss of the present invention
By 6.2ml n-butyl alcohol（27 moles of %）, 1.5ml ethanol（12 moles of %）, 2.9ml glacial acetic acids（19 moles of %）, 2ml water
（42 moles of %）, 0.012g 1,2,3-indantrione monohydrates（Per 1.0 grams of 1 liter of solution）Mix in chromatography cylinder, after stirring, obtain the group of the present invention
Compound.
With the aminoacid in the compositionss analysis fermentation liquid of the present invention
A) point sample：On chromatographic silica gel thin plate with pencil on the horizontal line of base 1cm（Point line-transect）Determine 13 points
（Origin）, each is put at a distance of about 3mm, point 4 same origins by 0.6 microlitre of fermentation liquid point containing aminoacid in step a)
On, per is dried up with hair-dryer for 1 time；
B) open up layer-colour developing：Step a) midpoints are had one end immersion of chromatographic silica gel thin plate of fermentation liquid equipped with of the present invention group
In the chromatography cylinder of compound, the liquid level of the present composition covers chromatography cylinder cap less than point line-transect, and the compositionss of the present invention are along layer
Analysis silica gel thin sheet opens up layer upwards.After 12 minutes, the compositionss of the present invention are opened up at the 2mm of thin plate upper edge, and layer is opened up in stopping；
C) chromatography silica gel thin sheet is taken out, with 70 DEG C of oven for drying 3 minutes, you can see the speckle of respective aminoacid.
As a result calculate
After measuring exhibition layer colour developing, each spot centers is to origin centre distance d1, then origin center is measured to solvent liquid level
Apart from d2, their Rf value is calculated, table 1 is as a result listed in.
Determine the Rf of aminoacid standard sample
Mark
Value
Aminoacid in the analysis method and result computational methods of aminoacid standard sample and fermentation liquid in above-mentioned fermentation liquid
Analysis method is similar with result method, and difference is to replace fermentation liquid with aminoacid standard sample.
By the Rf value of aminoacid in the fermentation liquid for measuring and the Rf of each aminoacid standard sample for determiningMarkValue is compared
The species of aminoacid in fermentation liquid, comparative result are listed in table 1.
1 measurement result of table
| d1(cm) | d2(cm) | Rf value | Amino acid classes |
| 0.50 | 3.95 | 0.13 | Lysine |
| 1.30 | 3.95 | 0.33 | Aspartic acid |
| 1.80 | 3.95 | 0.46 | Glutamic acid |
| 2.50 | 3.95 | 0.63 | Methionine |
| 2.80 | 3.95 | 0.71 | Isoleucine |
| 0.90 | 3.95 | 0.23 | Arginine |
| 0.60 | 3.95 | 0.15 | Histidine |
| 2.00 | 3.95 | 0.51 | Alanine |
| 2.65 | 3.95 | 0.67 | Tryptophan |
| 2.20 | 3.95 | 0.56 | L-Tyrosine |
| 1.45 | 3.95 | 0.37 | Serine |
| 3.00 | 3.95 | 0.76 | Phenylalanine |
| 1.60 | 3.95 | 0.41 | Threonine |
Embodiment 2：Prepare the test kit of the present invention
Raw material
It is same as Example 1.
Experiment equipment
15ml glass reagent bottle, purchased from Hangzhou Lai Baisi experimental facilitiess company limited.
Other experiment equipments are same as Example 1.
Prepare the test kit of the present invention
The test kit of the present invention includes：
Box 1：Including
14 bottle a, respectively 15ml glass reagent bottle, wherein containing 6.2ml n-butyl alcohol（27 moles of %）, 1.5ml ethanol
（12 moles of %）, 2.9ml glacial acetic acids（19 moles of %）With 2ml water（42 moles of %）Mixture,
14 bottle b, respectively 15ml glass reagent bottle, wherein containing 0.012g 1,2,3-indantrione monohydrates（Equivalent to per 1 liter of solution
1.0 gram）;
Box 2：Including
13 bottle c, respectively 15ml glass reagent bottle, wherein containing 10ml lysines, 10ml Radix Asparagi ammonia independently of one another
Acid, 10ml glutamic acid, 10ml methionine, 10ml isoleucine, 10ml arginine, 10ml histidine, 10ml alanine, 10ml colors
Propylhomoserin, 10ml L-Tyrosine, 10ml serines, 10ml Phenylalanine, the pure sample of 10ml threonine.
The bottle is respectively provided with the repeatable lid for covering tightly.
14 bottle a, 14 bottle b, 13 bottle c and lamellaes are provided in carton together.
With the aminoacid in the kit assay fermentation liquid of the present invention
Prepare fermentation liquid：The method for preparing fermentation liquid is same as Example 1.
With the aminoacid in the kit assay fermentation liquid of the present invention：
Material in above-mentioned 1 bottle a and in 1 bottle b is poured in chromatography cylinder, after mix homogeneously, is used in accordance with the following steps
Aminoacid in the kit assay fermentation liquid of the present invention：
A) point sample：On chromatographic silica gel thin plate with pencil on the horizontal line of base 1cm（Point line-transect）Determine 13 points
（Origin）, each is put at a distance of about 3mm, point 4 same origins by 0.6 microlitre of fermentation liquid point containing aminoacid in step a)
On, per is dried up with hair-dryer for 1 time；
B) open up layer-colour developing：Step a) midpoints are had one end immersion of chromatographic silica gel thin plate of fermentation liquid equipped with bottle a and bottle b
In material mixture chromatography cylinder in, the liquid level of the mixture covers chromatography cylinder cap, the mixture less than a point line-transect
Layer is opened up upwards along chromatographic silica gel thin plate.After 12 minutes, the mixture is opened up at the 2mm of thin plate upper edge, and layer is opened up in stopping；
C) chromatography silica gel thin sheet is taken out, with 70 DEG C of oven for drying 3 minutes, you can see the speckle of respective aminoacid.
As a result calculate：As a result computational methods are same as Example 1.Result of calculation is identical with table 1.
Determine the Rf scale values of aminoacid standard sample：The analysis method and result computational methods of aminoacid standard sample with it is upper
The amino acid analysis method stated in fermentation liquid in fermentation liquid is similar with result method, and difference is, with the present embodiment box 2
Aminoacid standard sample replace fermentation liquid.
By the Rf value of aminoacid in the fermentation liquid for measuring and the Rf of each aminoacid standard sample for determiningMarkValue is compared
The species of aminoacid in fermentation liquid, comparative result are identical with table 1.
It should be understood that the needs in order to adapt to large scale test, the above-mentioned amount of the present composition can be put in proportion
Greatly, as long as compositionss comprising material described in the present composition and amount ratio is in the range of the present invention is referred to, belong to this
The claimed scope of invention.
Claims (23)
1. a kind of method of use compositionss analysis of amino acid, the compositionss are the mixing containing alcohol, acid, water and 1,2,3-indantrione monohydrate
Thing, the alcohol are the alcohol mixture of n-butyl alcohol and ethanol, the mole ratio of n-butanol of the alcohol mixture mixing:Ethanol is 2/1-7/
2, the acid is glacial acetic acid, and
Wherein, the amount of the alcohol is 30-40 mole of %, and the amount of the acid is 18-25 mole of %, and the amount of the water rubs for 40-45
You are %, and the amount sum based on the alcohol, acid and water is 100 moles of % meters；The amount of the 1,2,3-indantrione monohydrate be per 0.8-5 gram of 1 liter of solvent,
And wherein described aminoacid include lysine, aspartic acid, glutamic acid, methionine, isoleucine, arginine, histidine,
Alanine, tryptophan, L-Tyrosine, serine, Phenylalanine, threonine.
2. claim 1 methods described, wherein the mixture containing alcohol, acid, water and 1,2,3-indantrione monohydrate is by alcohol, acid, water and indenes three
Ketone is formulated.
3. claim 2 methods described, wherein the mixture containing alcohol, acid, water and 1,2,3-indantrione monohydrate is by alcohol, acid, water and solid
1,2,3-indantrione monohydrate is formulated.
4. a kind of method of use kit assay aminoacid, the test kit are included selected from one of following container：
Accommodate the container of alcohol, acid, water and 1,2,3-indantrione monohydrate mixture；
Accommodate alcohol, the container of acid and water and accommodate the container of 1,2,3-indantrione monohydrate；Or
Alcohol, acid, the container of water are accommodated respectively and accommodate the container of 1,2,3-indantrione monohydrate；Or
Accommodate respectively alcohol, acid, optional two kinds in water of container and the container containing another material in alcohol, acid, water with
And accommodate the container of 1,2,3-indantrione monohydrate；
The alcohol is n-butyl alcohol and the alcohol mixture of ethanol, the mixing mole ratio of n-butanol of the alcohol mixture:Ethanol is 2/1-
7/2, the acid is glacial acetic acid, and
Wherein, the amount of the alcohol is 30-40 mole of %, and the amount of the acid is 18-25 mole of %, and the amount of the water rubs for 40-45
You are %, and the amount sum based on the alcohol, acid and water is 100 moles of % meters；The amount of the 1,2,3-indantrione monohydrate be per 0.8-5 gram of 1 liter of solvent,
And wherein described aminoacid include lysine, aspartic acid, glutamic acid, methionine, isoleucine, arginine, histidine,
Alanine, tryptophan, L-Tyrosine, serine, Phenylalanine, threonine.
5. the method described in claim 4, the test kit also include flat board and/or the container containing control sample.
6. the method described in claim 4, wherein the mixture containing alcohol, acid, water and 1,2,3-indantrione monohydrate is by alcohol, acid, water and solid
Body 1,2,3-indantrione monohydrate is formulated, or, what is accommodated in the container for accommodating 1,2,3-indantrione monohydrate is solid 1,2,3-indantrione monohydrate.
7. compositionss described in any one of claim 1-3 or test kit described in any one of claim 4-6 are used for analysis of amino acid
Purposes, wherein the aminoacid include lysine, aspartic acid, glutamic acid, methionine, isoleucine, arginine, group ammonia
Acid, alanine, tryptophan, L-Tyrosine, serine, Phenylalanine, threonine.
8. the purposes described in claim 7, wherein the aminoacid is the aminoacid in fermentation liquid.
9. the purposes described in claim 8, wherein the fermentation liquid is the aqueous solution obtained after fermented product dilute with water.
10. the purposes described in claim 9, wherein the fermentation liquid is the fermentation liquid that 1-100 times of fermented product dilute with water is obtained.
Purposes described in 11. claim 9, wherein the fermented product is with selected from glucose, Lactose, sucrose, maltose, Semen Maydiss
Oil, the sugar of amylum hydrolysate of the sugar are for substrate and with selected from Deshi Lactobacilluss (Lactobacillus delbrueckii), De Shi breast bars
Bacterium subspecies bulgaricus, the microorganism in Deshi Lactobacilluss Lai Ku South Asia kind (L.leichnanii) carry out cultivation and fermentation acquisition.
A kind of 12. methods of analysis of amino acid, methods described comprise the steps：
A) point sample：By the sample spot containing aminoacid on flat board；
B) open up layer-colour developing：One end that point has the flat board containing aminoacid sample is placed in into the mixing of containing alcohol, acid, water and 1,2,3-indantrione monohydrate
In thing, the compositionss or right that the mixture of the containing alcohol, acid and water and 1,2,3-indantrione monohydrate is derived from described in any one of claim 1-3 will
The test kit described in any one of 4-6 is sought,
Wherein, the aminoacid include lysine, aspartic acid, glutamic acid, methionine, isoleucine, arginine, histidine,
Alanine, tryptophan, L-Tyrosine, serine, Phenylalanine, threonine.
13. the method described in claim 12, wherein described in step a), flat board is lamellae.
Method described in 14. claim 12, wherein described in step a), the sample containing aminoacid is aminoacid standard sample,
And the solution to be measured containing aminoacid.
Method described in 15. claim 14, wherein the solution to be measured containing aminoacid is the fermentation liquid containing aminoacid.
Method described in 16. claim 15, wherein the fermentation liquid containing aminoacid be fermented product dilute with water after obtain
Aqueous solution.
Method described in 17. claim 16, wherein the fermentation liquid containing aminoacid is 1-100 times of fermented product dilute with water
The fermentation liquid for obtaining.
Method described in 18. claim 16 or 17, wherein the fermented product with selected from glucose, Lactose, sucrose, maltose,
Semen Maydis oil, the sugar of amylum hydrolysate of the sugar are for substrate and with selected from Deshi Lactobacilluss (Lactobacillus delbrueckii), De Shi
Lactobacilluss subspecies bulgaricus, the microorganism in Deshi Lactobacilluss Lai Ku South Asia kind (L.leichnanii) carry out cultivation and fermentation and obtain
.
Method described in 19. claim 12, wherein described in step a), point sample step is repeatedly carried out.
Method described in 20. claim 19, wherein also including the step of being dried after the completion of each point sample.
The liquid of the mixture of the alcohol, the acid and water and 1,2,3-indantrione monohydrate in the method described in 21. claim 12, wherein step b)
Face is not higher than the sample spot on flat board.
Method described in 22. claim 12, wherein the method for the analysis of amino acid also includes：C) containing alcohol, acid, water and indenes are treated
After the completion of the mixture of triketone opens up layer-colour developing on the flat board, flat board is taken out, which is dried, it is seen that various aminoacid
Speckle.
23. the method described in claim 22, wherein the method for the analysis of amino acid also includes：D) will derive from flat board c) with
The flat board of standard sample compares.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201410048846.2A CN103823014B (en) | 2014-02-12 | 2014-02-12 | Compositionss, test kit and application thereof and with its analysis of amino acid method |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201410048846.2A CN103823014B (en) | 2014-02-12 | 2014-02-12 | Compositionss, test kit and application thereof and with its analysis of amino acid method |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN103823014A CN103823014A (en) | 2014-05-28 |
| CN103823014B true CN103823014B (en) | 2017-03-29 |
Family
ID=50758177
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201410048846.2A Active CN103823014B (en) | 2014-02-12 | 2014-02-12 | Compositionss, test kit and application thereof and with its analysis of amino acid method |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN103823014B (en) |
Families Citing this family (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104360005B (en) * | 2014-11-05 | 2017-02-15 | 无锡晶海氨基酸有限公司 | Method for quickly assessing quality of industrial corn steep liquor applied to fermentation of amino acid |
| CN104459008B (en) * | 2014-12-09 | 2016-05-11 | 山东拜尔检测有限公司 | A kind of quick detection kit of protein |
| CN117607334A (en) * | 2023-11-27 | 2024-02-27 | 南方医科大学南方医院 | Diabetes skin lesion risk screening kit and screening method |
Family Cites Families (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| RU2030741C1 (en) * | 1991-07-16 | 1995-03-10 | Научно-производственное товарищество "Протеин ПАВ" | Method for qualitative analysis of sorption activity of aminoacids |
| RS52068B (en) * | 2005-12-07 | 2012-06-30 | Basilea Pharmaceutica Ag. | Useful monobactam antibiotics |
| CN102125598B (en) * | 2010-01-12 | 2012-09-26 | 桂林三金药业股份有限公司 | Quality control method of watermelon frost |
| CN103063796A (en) * | 2011-10-24 | 2013-04-24 | 青海金本生物科技实业股份有限公司 | Detection method of dry sheep placenta |
| CN103217502A (en) * | 2013-04-15 | 2013-07-24 | 浙江大德药业集团有限公司 | Detection method of capsule for reinforcing kidney and warming yang |
-
2014
- 2014-02-12 CN CN201410048846.2A patent/CN103823014B/en active Active
Also Published As
| Publication number | Publication date |
|---|---|
| CN103823014A (en) | 2014-05-28 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Hofman et al. | High-throughput quantification of hydroxyproline for determination of collagen | |
| Xiao-wei et al. | Detection of meat-borne trimethylamine based on nanoporous colorimetric sensor arrays | |
| CN103823014B (en) | Compositionss, test kit and application thereof and with its analysis of amino acid method | |
| Wu et al. | Application of SYBRgreen PCR and 2DGE methods to authenticate edible bird's nest food | |
| CN103852460B (en) | Based on the method that how residual the magnetic nano fluorescent sensor detection of antibiotics of aptamers is | |
| CN105092571A (en) | Method for quickly detecting application performance of titanium dioxide of oil paint systems | |
| CN101629953A (en) | On-site detection immuno-chip and preparation method thereof and application | |
| CN104498597A (en) | Multiplex PCR rapid detection method for common contraindicant ingredients in muslem food | |
| CN104561271A (en) | Visual DNA (deoxyribonucleic acid) chip kit and method for detecting multiple animal-derived components | |
| CN107064286A (en) | The method of Gram-positive and negative bacterium Mass Spectrometer Method kit and Rapid identification | |
| Rühmann et al. | Automated modular high throughput exopolysaccharide screening platform coupled with highly sensitive carbohydrate fingerprint analysis | |
| CN207623349U (en) | A kind of porcine pseudorabies virus antibody assay kit | |
| CN102798560A (en) | Clenbuterol matrix standard substance and preparation method thereof | |
| CN104374907B (en) | A kind of indirect competitive enzyme-linked aptamers detects the method for Determination of oxytetracycline residues | |
| Karlsson et al. | Quantitation of proteoglycans in biological fluids using Alcian blue | |
| CN103267745B (en) | A kind of endotoxin MIP-SPR chip, preparation method and its usage | |
| Lee et al. | Sodium dodecyl sulfate polyacrylamide gel electrophoresis for direct quantitation of protein adsorption | |
| CN104931544A (en) | Method of producing microarray aptamer sensor used for antibiotic residue detection | |
| Lee et al. | Solid-phase colorimetric method for the quantification of fucoidan | |
| CN107024459A (en) | Neomycin molecular engram quanta polymer and preparation method for detecting neomycin | |
| CN203965445U (en) | A kind of dynamic color method limulus kit for endotoxin detection | |
| CN204188621U (en) | A kind of dynamic color method detecting limulus kit for glucan in fungus | |
| CN110320204A (en) | The colorimetric detection method of yapamicin relict in a kind of milk | |
| EP3546942B1 (en) | Biochip | |
| CN104152548B (en) | A kind ofly detect biochip, detection kit and the detection method of producing flavacin mould |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |